Atherosclerosis is a complex progressive disease involving intertwined biological mechanisms. We aimed to identify miRNA expression dynamics at the early stages of atherosclerosis using a large swine model . A total of 18 female pigs; 9 familial hypercholesterolemic (WMS-FH) and 9 normal control swine (WMS-N) were studied. miRNA sequencing was performed on plasma cell-free RNA at 3, 6, and 9 months of age. RT-qPCR validated DE miRNAs in a new cohort of animals (n = 30) with both sexes. Gene ontology and mRNA targets for DE miRNAs were identified. In vivo multimodality imaging and histopathology were performed to document the presence of atherosclerosis at termination. 20, 19, and 9 miRNAs were significantly DE between the groups at months 3, 6, and 9, respectively. Most DE miRNAs and their target genes are involved in human atherosclerosis development. Coronary atherosclerosis was documented in 7/9 WMS-FH pigs. Control animals had no lesions. miR-138, miR-152, miR-190a, and miR-196a showed a significant diagnostic power at month 3, whereas miR-486, miR-126-3p, miR-335, and miR-423-5p were of significant diagnostic power at month 9. In conclusion, specific DE miRNAs with significant discriminatory power may be promising biomarkers for the early detection of coronary atherosclerosis. Other biomarkers such as serum C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα) have shown promising results in predicting future cardiac events4. However, they have not been widely adopted due to their high biological variability in specific patient populations and clinical subsets2.Atherosclerosis is a chronic condition that slowly develops over decades. At the early stages of development, disease detection is particularly important to delay the progression of disease. Lipid biomarkers  have historically been the standard of care to screen high-risk patients. However, these biomarkers have insufficient predictive value especially amongst individuals with normal cholesterol levels8 and are well-suited to be used as biomarkers for atherosclerosis detection. Human studies using circulating miRNAs in atherosclerosis have primarily included patients in which the disease is already present, in an advanced stage or become symptomatic 10. Longitudinal studies aimed to characterize the dynamics of circulating miRNA changes at the early stages of disease development are limited. In the present work, we used a well-established model of atherosclerosis 15 to study; (a) the temporal dynamics of miRNA changes, (b) the miRNA signatures specific to early atherosclerosis development and (c) the diagnostic power of these biomarkers for the detection of the disease. Plasma miRNA levels were assessed at three different time points  and differentially expressed (DE) miRNAs were compared between WMS-FH and genetically related WMS-N controls. The presence or absence of disease was confirmed by multi-modality imaging and histo-pathology evaluation at 12 months. The diagnostic power of each miRNA in discriminating between the presence and absence of atherosclerosis was calculated and gene ontology (GO) and mRNA targets of DE miRNAs were identified to understand the potential biological mechanisms involved in atherosclerosis development at this early stage. The methodology followed in this work is shown in  play a role in cardiovascular disease developmentp < 0.001). A certain level of variability in total cholesterol levels is commonly seen in WMS-FH model . Cholesterol levels stabilized at 6 months and did not change much by 9-months . There were no significant changes in cholesterol levels in the WMS-N control group.The characteristics of the animals used for small RNA sequencing are summarized in supplemental Table del Fig. . Total cAll animals underwent multimodality imaging with angiography Fig. A, IVUS B, and OCA summary of the immunohistochemistry findings by animal is presented in Table The coronary segments exhibiting atherosclerotic changes had easily discernible intimal hyperplasia. CD61/CD163-1 positive staining cells were common within these regions and the distribution was not uniform as the density of positive cells varied throughout the intimal hyperplasia around the circumference of the affected artery. The CD61/CD163-1 positive staining cells appeared to be at least slightly more prominent near the IEL as compared to regions near the luminal surface. Commonly, the CD61/CD163-1 positive staining extended into the tunica media in focal areas contiguous with regions of positive staining in the intimal hyperplasia. In some of the atherosclerotic segments, segmental areas of minimal to mild hypercellularity of the adventitia contained CD61/CD163-1 positive cells, which were in a higher density than the native, positive staining cells observed in regions of normal adventitia Fig. .Figure 4p < 0.1. A total of 20, 19, and 9 DE miRNAs were identified for months 3, 6, and 9, respectively. In addition, some DE miRNAs were present in at least two-time points . Moreover, DE miR-194b-5p showed expression differences between females and males (p = 0.036) at month 9, where females exhibited higher expression for this miRNA. Similar to miRNA sequencing results, upregulated miR-206, downregulated miR-138, and miR-126-3p were also validated at month 6. No statistically significant differences between WMS-FH and WMS-N were found for miR-138 at month 3. Supplemental Table We validated a subset of DE miRNAs in a new cohort of animals. Among the tested miRNAs, miR-194b-5p was differentially expressed between WMS-FH and WMS-N at 3, 6, and 9 months of age; however, the RT-qPCR showed a significantly higher expression in WMS-FH at months 6 and 9 but not at month 3. Also, miR-194b-5p expression increased with age  to study the DE of circulating miRNAs over a period of 9 months and correlated these changes with the histological presence of atherosclerosis. The study findings show that (a) atherosclerosis was diagnosed by in vivo multimodality imaging and confirmed by histopathology in 7/9 (78%) of the WMS-FH and in none of the animals in the control group, (b) several DE miRNAs were identified at multiple time points and few remained DE overtime, (c) some of these DE miRNAs showed decent discriminatory power for the diagnosis of coronary artery atherosclerosis and (d) the gene targets and biological processes influenced by these miRNAs provided insight into their possible biological relevance in the context of atherosclerosis development.Early atherosclerosis detection is a critical step towards the prevention of disease progression and acute plaque destabilization. Several studies have proposed the use of circulating miRNAs as biomarkers to diagnose the presence of atherosclerosis in humansOur study demonstrated that circulating miRNA profiles start to differ between WMS-FH and WMS-N at very early stages of atherosclerosis development and continue to change over time. Only a handful of miRNAs remained DE over time, demonstrating the complexity and variability of the biological processes involved in atherosclerotic lesions. Our results indicate that DE miRNAs undergo dynamic changes in the early stages of atherosclerosis and continue to be dysregulated in the advanced stages of disease development. These miRNAs appear and reappear over time depending on the biological state of the disease and the involved mechanisms. This is supported by the detection of DE miR-194b-5p at months 3, 6, and 9 and other miRNAs that were captured only at one time point in the present work.20. In contrast, miR-146a and miR-146-5p were downregulated in the WMS-FH at 3 months. Specifically, miR-146-5p appears to have an atheroprotective effect by preventing foam cell formation through the downregulation of NF-κB and TRAF6 expression22.The nature of the DE miRNAs changes seen over time supports some of the well-described biological mechanisms involved in atherosclerosis development. At 3 months, many of the DE miRNAs regulate biological processes involving cell adhesion, migration, and proliferation. Regulation of cellular cholesterol clearance and foam cell formation were predominant at this timeframe. Among the DE miRNAs found in our study, miR-7, miR-144, miR-146a-5p, and miR-24-3p regulate foam cell formation. miR-7 and miR-144 were upregulated and promote foam cell formation by directly downregulating genes involved in cholesterol efflux (STK11 and TR4)29 and SCF23. Another downregulated miRNA in WMS-FH is miR-182 which was shown recently to inhibit vascular smooth muscle cell proliferation24. Similarly, miR-24 was shown to inhibit endothelial cell proliferation25. Moreover, reduced expression of miR-24 resulted in increased MMP14 expression and lesion size in mice26.DE miRNAs involved in angiogenesis and smooth muscle cell proliferation were predominant at 6 months. miRNAs involved in the regulation of smooth muscle cell dynamics, including regulation of phenotypic switching, actin cytoskeleton organization and activation of protein kinase B activity were particularly prominent. In our study, miR-23b, miR-206, miR-204, miR-1, and miR-34c, known to regulate smooth muscle cell proliferation, were differentially expressed. miR-23b and miR-34c were downregulated in WMS-FH and have been shown to tightly regulate SMC proliferation and prevent neointima formation by targeting LRP628. Therefore, the consistent upregulation of this miRNA indicates another lacking mechanism of atheroprotection in WMS-FH that is persistent and spans a long period. However, the proatherogenic role of miR-194b-5p remains to be investigated.The only miRNA upregulated across the three time points in the WMS-FH was miR-194b-5p. This miRNA targets IL-10 and is involved in its negative regulation  cascade, and the phosphatidylinositol-3-kinase (PI3K) signaling pathway41 and targeting key mediators such as VCAM-1, IL-6, and TNF-α42. In humans, miR-126-3p expression levels were shown to distinguish between individuals with and without coronary atherosclerosis43. Moreover, this miRNA was significantly lower in patients with coronary artery atherosclerosis compared to healthy patients44. miR-423-5p does not have a well-described biological function or role in atherosclerosis.There were no DE miRNAs with discriminatory power at 6 months. At 9 months miR-486-5p, miR-335-5p, miR-126-3p, and miR-423-5p had the highest diagnostic discriminatory power to detect coronary atherosclerosis. miR-486-5p was upregulated and reported to target HAT1, which then downregulates ABCA1, hence, affecting cholesterol efflux47. An unexplored mechanism captured in our data is the regulation of lamellipodium assembly that appears to be targeted by miR-196a. Lamellipodium assembly plays a crucial role in cell migration48, a process that is heavily involved in atherosclerosis. Also, our findings suggest an association between miR-138 and K63-linked ubiquitination, a regulatory mechanism associated with inflammation, cell survival, and oxidative stress49. In addition, miR-335 was shown to regulate the proliferation and smooth muscle cells phenotypic switching through direct modulation of SP150. The relevance of this mechanism in atherosclerosis is yet to be established and warrants further investigation. A remarkably interesting mechanism is the negative regulation of amyloid beta clearance that appears to be associated with DE miR-7. Aberrant deposition and clearance of amyloid from the subintima have been linked to inflammation and the promotion of atherosclerosis51. Also, miR-138 plays a role in the regulation of osteoblast differentiation52, suggesting that vascular calcification starts early in the process of atherosclerosis development.miR-196a is the miRNA with the highest discriminatory power and earliest detection (month 3) in the present study. Few studies have explored its role in atherosclerosis development53. Furthermore, aberrations in epigenetic marks have been linked to atherogenesis56. Some of the miRNAs found in this study  did not share similarities with any known miRNA human, mice, rat, rabbits, or non-human primate sequences currently available in miRBase. The uniqueness of these miRNAs suggests that they may be swine-specific. However, these miRNAs may also be novel ones that are yet to be identified. Nonetheless, these miRNAs deserve further investigations to assess their role in atherosclerosis.Finally, several DE miRNAs were found to target epigenetic modifiers such as DNMT1, DNMT3A, and EP300, suggesting the presence of alterations in epigenetics mechanisms between WMS-FH and WMS-N. Alterations in epigenetic marks result in aberrant gene expression and phenotypesOne limitation of the present work is that GO search was carried out for those miRNAs similar in sequence to human miRNAs. Another limitation was the absence of live imaging in animals at each studied time point to confirm the absence of the disease in early life. However, WMS-FH swine develop lesions in the arteries around 1-year-old when the animals are fed the normal diet used in this work. Also, the work focused on early atherosclerosis, which is confirmed with OCT imaging and histopathology that indicates the absence of advanced stages of the disease in FH animals. Also, our study is limited to miRNA profiles at months 3, 6 and 9 of age. Profiling earlier time points than month 3, could potentially provide earlier biomarkers for atherosclerosis detection. However, we have opted for the time points for the following reasons. (1) WMSH FH show a peak in cholesterol levels around month 6 of age. (2) The total cholesterol levels stabilize around month 9. (3) Month 3 was selected a time point that allows for similar intervals of time for profiling. Additionally, we observe lesions in this model typically around the age of 12 months with no lesions observed at month 9, 6 or 3.The findings above demonstrate that circulating miRNAs expression differs between WMS-FH and WMS-N, and these differences can be identified in animals’ early life before the manifestation of atherosclerosis. Additionally, these miRNAs are involved in mechanisms governing the pathophysiology of the disease, further supporting the evidence of early onset before disease manifestation; hence, these miRNAs are promising biomarkers for early detection. Specifically, in WMS-FH, atherosclerosis can be detected at 3 months of age using miR-138, miR-152, miR-190a, and miR-196a. While miRNAs miR486, miR-126-3p, miR-335, and miR-423-5p can detect the disease at month 9 of age.Guide for the Care and Use for Laboratory Animals57 and the Animal Welfare Act58. All methods herein are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). The methodology followed in the present study is summarized in Fig. The study was approved by the Institutional Animal Care and Use Committees of the the University of Wisconsin-Madison  and CRF Skirball Center of Innovation . All animals received standard care in compliance with the Two cohorts of the WMS-FH and WMS normal (WMS-N) were selected for this study. WMS-N pigs have the same genetic background as WMS-FH; however, these swine are normocholesterolemic with a completely functional LDLR. Cohort 1 had nine WMS-FH  and nine WMS-N . In the second cohort, 15 WMS-FH  and 15 WMS-N  were included. Animals were randomly selected and housed with the main herd at the Swine Research and Teaching Center (SRTC) at the University of Wisconsin-Madison throughout the duration of the study. All animals were fed the same diet formulated to meet the nutritional requirements of swine  was performed using QAngio XA SoftwareTM 7.1.14.0 . A contrast-filled catheter was used for calibration. The minimum lumen diameter (MLD) was obtained from a single view with the lowest measurement, while the reference vessel diameter (RVD) was automatically calculated. Percent diameter stenosis (%DS) was calculated from the MLD and the RVD.Intravascular Ultrasound (IVUS) pullback images were generated  and analyzed with a commercially available software . Luminal- and vessel areas were measured, and the plaque burden and plaque volume were calculated. Optical Coherence Tomography (OCT) images were obtained using the ILUMIEN OPTIS imaging system . Qualitative analyses were performed at 1-mm intervals with the commercial software . Cross-section lumen, vessel areas and intimal thickness were measured, and percent area stenosis was calculated as (vessel area-lumen area)/vessel area * 100%.Whole blood was collected longitudinally at 3, 6, and 9 months from each animal in 8 mL K3 EDTA tubes . Blood tubes were centrifuged at 4 °C for 10 min to separate plasma. Next, collected plasma was centrifuged using the same settings to remove any cellular debris. Samples were then preserved in 1.5 mL tubes at − 80 °C until needed. The three-time points were selected based on the cholesterol profiles of WMS-FH pigs. Month 3 was selected as an early time point before serum cholesterol levels peak and stabilize. Total cholesterol then peaks around month 6 and stabilizes around month 9.59. To correct for this effect, we measured hemolysis in plasma samples by assessing absorbance at A414 using NanodropONE spectrophotometry . For each sample, the average of at least three measurements was calculated. The A414 absorbance values were used as a covariate in statistical analysis to assess the differential expression of miRNAs. Total cholesterol levels were measured in plasma samples using ELISA Cholesterol Assay Kit—HDL and LDL/VLDL  following the product’s manual. Plasma samples from WMS-FH were diluted (twofold) so that colorimetric readings fell within the range of the cholesterol standard curve.Studies have shown that hemolysis considerably affects the levels of cell-free miRNAsCell-free RNA (cf-RNA) was extracted from plasma samples using Quick-cfRNA Serum & Plasma Kit . Briefly, plasma samples were centrifuged at 12,000 g for 15 min to remove cellular debris, then 400 uL of plasma was digested for 2 h at 37 °C using proteinase K. After digestion, the remaining steps were followed as described in the manufacturer’s manual. To obtain concentrated samples, we eluted the column content in 10-uL of nuclease-free water. For biological validation samples used in RT-qPCR , cel-miR-39 mimic  was spiked in each sample after 2 h of digestion to assess extraction efficiency and consistency across samples.60 was used to assess sequencing quality. We performed bioinformatics analysis using OASIS for small RNA sequencing pipeline61. Briefly, trimmed reads were aligned to the swine miRNA database and reference genome (Sus Scrofa ssc3) using STAR62. Next, using DESeq263, the obtained mapped raw reads were normalized, and differential expression was performed following the negative binomial generalized linear regression model. The regression model included hemolysis in plasma samples, weight of animals and grouping (WMS-FH vs. WMS-N) for each time point . Time course analysis to detect changes in miRNA expression within individual animals and between groups included the age of animal as an additional variable . Only miRNAs with adjusted p value < 0.1 were considered significantly differentially expressed between WMS-FH and WMS-N.Small RNA sequencing was performed by RealSeq-Biosciences . Library preparation was carried out using the RealSeq-Biofluids library prep kit . Libraries were then sequenced on a NextSeq 500 v2 High-Output with a read length of 75 bp in one direction (single read). Both raw and trimmed fastq files were obtained from RealSeq-Biosciences. For each file, FastQC16 to check for sequence similarity. Human miRNAs that are (> 90%) similar in sequence to the swine miRNAs were used to search for mRNA targets available in the database of validated miRNA-target interactions (miRTarBase)64. GO terms for each miRNA were obtained using miRBase database65.To assess the biological function of the differentially expressed miRNAs, we predicted the miRNA targets and miRNA functions via Gene Ontology (GO). Swine differentially expressed miRNA sequences were blasted against the human miRNAs using the miRBase database66. miR-17-3p showed high stability in all sequenced samples. cf-RNA extraction was repeated for samples showing variability of cel-miR-39 and UniSp6 expression. The common base method67 was used to assess differential expression, where each well of the RT-qPCR assay efficiency was incorporated in the final calculation of the ΔΔCT method68.We selected a subset of DE miRNAs for validation in a new cohort of animals. Selection criteria were based on expression pattern (up/downregulation), early time point of detection (month 3 or 6), and detection at multiple time points . A total of 30 samples (cohort 2) were used to validate DE miRNAs. For each plasma sample, 2-uL of extracted cf-RNAs were used in cDNA synthesis with MIRCURY LNA RT kit (QIAGEN). Additionally, each cDNA reaction was spiked in with UniSp6 to assess a consistent cDNA synthesis across all samples. MIRCURY LNA assay primers were obtained from QIAGEN. MIRCURY LNA SYBR Green assay kit (QIAGEN) was used to assess the expression of miRNAs. Also, two miRNA endogenous controls (bta-miR-93 and miR-17-3p) were used to normalize expression results. The endogenous controls were selected based on the stability of multiple miRNAs using NormfinderRepresentative samples of the proximal to mid regions of the right coronary artery (RCA) and/or the left anterior descending coronary artery (LAD) were taken for histology following fixation consisting of perfusion of the coronary vasculature with 1 L of 10% neutral buffered formalin after initial clearance of blood with 1 L of normal saline and then additional immersion fixation in 10% NBF for a minimum of 24 h. The arterial segments were processed by routine methods and embedded in paraffin. The resulting paraffin blocks were cut at 5-μm thickness and immunohistochemically stained for CD68 and CD163-1 and examined for the presence, relative amount, and distribution of positive staining. Monoclonal antibodies for CD68 (M1-type macrophage marker) and CD163-1 (M2-type macrophage markers) were used simultaneously with the secondary antibody (mouse-on-FARMA-HRP (horseradish peroxidase) and diaminobenzamide (DAB) was used as a chromogen) detecting both primary antibodies with no differentiation between the two; therefore, the immunohistochemical assay was designed to detect all macrophages within the examined tissue without differentiating between the 2 subtypes (panmacrophage IHC). Lymph nodes from swine served as a positive control. Sections of the same lymph nodes were stained with the same procedure using normal mouse IgG as the primary antibody to serve as a control for the assessment of nonspecific staining. The IHC-stained sections were evaluated for the presence of the DAB chromogen. The amount of positive staining was semiquantitatively scored using standard methods. The control section from the full staining protocol showed intensely positive staining cells in regions of the lymph nodes consistent with macrophage populations; therefore, the IHC assay performed well in detecting tissue macrophages. The sections were counterstained with H&E. The primary control section showed no discernible staining; therefore, there was no discernible nonspecific binding by the secondary antibody. The IHC stain employed in this investigation was not designed to allow distinction of the details of tissue architecture, as the tissue stains were only the DAB chromogen of the CD61/CD163-1-positive cells and a hematoxylin counterstain. However, the presence of certain specific features including intimal thickening/hyperplasia, cellular density, and characteristics consistent with atherosclerosis could be distinguished, and their association with the CD61/CD163-1 positive staining was noted when present.69. First, animals were categorized into “healthy” and “with disease” based on imaging and histo-pathology results. Only significantly DE miRNAs obtained with DESeq2 from each time point were used in this step. Next, we used normalized sequencing reads for each miRNA to measure the area under the curve (AUC), the confidence interval, and significance level. Due to the small sample size, we determined those miRNAs with AUCs that show a statistical power of 0.95 and 0.8 with α = 0.05.To determine the diagnostic power of miRNAs, we performed a discriminatory power analysis using the ROC curves of the pROC R package70. Finally, the statistical significance of the AUC was estimated using power.roc.test function in pROC package69.A two tailed t test was used to assess significant differences in animal characteristic between groups. For miRNA sequencing data, DE miRNAs and statistical significance were determined using a negative binomial regression model that included the following covariables: hemolysis, weight, and group (WMS-FH vs. WMS-N). For RT-qPCR gene expression, a general linear regression model was used to determine the statistical significance in miRNA differences between WMS-FH and WMS-N with animal weight, plasma hemolysis, and sex of animals as variables using the R programming languageSupplementary Information.


Positron emission tomography and computed tomography (PET-CT) is currently recommended in evaluating the treatment response after (chemo)radiotherapy ([C]RT). In the larynx, post-treatment changes and physiological uptake make image interpretation more challenging compared to other head and neck sites. Previous research has not addressed imaging factors specifically in the larynx that would help in distinguishing the residual disease and explain the unique challenges of that anatomic area. The study cohorts are small and heterogenous. Our objective was to investigate the ability of PET-CT in diagnosing local residual laryngeal carcinoma, and to uncover imaging factors that could be used in differentiating the residual disease from post-treatment and physiological changes. In the same study cohort, we also aimed to uncover prognostic factors for local residual or recurrent disease.Our retrospective study cohort included 73 patients with T2-T4 laryngeal carcinoma undergoing (C)RT with curative intention, and post-treatment non-contrast-enhanced PET-CT at 2–6 months. Findings were compared between local residual and non-residual disease. Local residual disease was defined as a persistent tumor growth with no evidence of remission in between, confirmed by biopsy, and evident within 6 months from the end of RT. PET-CT was evaluated using a 3-step scale: negative, equivocal, and positive.max of over 4.0 (p < 0.001). CT showed a persistent mass at the primary tumor area in 56% of residuals, and in 23% of non-residuals (p > 0.05). By combining SUVmax>4.0 and mass, specificity improved to 91%.Nine (12%) had a local residual tumor and 11 (15%) developed local recurrence, based on the biopsy. The median follow-up of surviving patients was 64 months . In univariate analysis, primary tumor diameter greater than 2.4 cm , and vocal cord fixation were prognostic for local residual or recurrent disease. Sensitivity, specificity, PPV, and NPV were 100%, 75%, 36%, and 100%, respectively, when the equivocal interpretation was grouped with the positive interpretation. All local residuals, and 28% (18/64) non-residuals, had a primary tumor area SUVmax over 4.0. The combination of SUVmax over 4.0 and mass on CT increased specificity, but the sensitivity was low.NPV of post-treatment PET-CT in laryngeal carcinoma is high, but equivocal and positive results have low PPV and require further diagnostics. All local residuals had SUV Radiotherapy (RT) and chemoradiotherapy (CRT) are important function-preserving treatment options for laryngeal carcinoma . After sOur primary aims are to investigate the ability of post-treatment PET-CT in uncovering residual disease within the larynx, and to document the spectrum of imaging findings so that they could be used more reliably in the treatment response evaluation. Our secondary aim was to uncover pre-treatment patient or imaging characteristics that are prognostic for local residual or recurrent disease.max) could not be retrospectively measured due to technical difficulties. The final study cohort consisted of 73 patients, including 71 squamous cell carcinomas, 1 adenocystic carcinoma, and 1 sarcomatotic carcinoma. Table For this retrospective study we included patients with T2-T4 laryngeal carcinoma, diagnosed at Helsinki University Hospital Department of Otorhinolaryngology – Head and Neck Surgery between 2006 and 2018, and who underwent (C)RT with curative intention, and had post-treatment PET-CT examination at 2–6 months. The patients were retrieved from the hospital database using the International Classification of Diseases code C32. We excluded patients with previous HNC. Further, two patients were excluded because the maximum standard uptake values . The follow-up time frame for patients ranged from 28 to 174 months , or until death. Of all 35 surviving patients at the end of our evaluation period, 33 (94.3%) had a minimum follow-up of 3 years.Multidisciplinary tumor board meetings reviewed the diagnostics and staging for all patients and gave treatment recommendations according to national guidelines. Altogether, 60 (82%) patients were treated with CRT and 13 (18%) with RT alone. The mean RT dose was 69.6 Gy (range 60–72). The majority of patients received Cisplatin 40 mg/mLocal remission was defined as imaging without any evidence of disease, or negative biopsies from the primary tumor area. Definition for local residual disease was a persistent tumor with no proof of remission, confirmed with biopsy, and evident within 6 months from the end of radiotherapy. Local recurrence was defined by a new tumor growth confirmed with biopsy after the patient’s previous remission.Pre-treatment scans were retrospectively re-evaluated by a radiologist (H.S.) specialized in head and neck imaging. Altogether, 44 patients had MRI, 25 had CT, and 1 had PET-CT. One patient had both CT and MRI, one patient had MRI and PET-CT and one patient had CT and PET-CT.After treatment, the patients were followed up with regular clinical examinations and PET-CT at 2–6 months. The median time from the end of treatment to the first follow-up PET-CT was 94 days (range 68–176 days). Post-treatment PET-CT images were retrospectively re-evaluated by a radiologist specialized in head and neck imaging (H.S.) and briefed to the PET-CT interpretation by a nuclear medicine specialist (J.S.). The radiologist was blinded to the original report and to the results of treatment. When the interpretation differed from the original report assessed by a nuclear medicine physician and a radiologist, a consensus was made together with another head and neck radiologist (A.M.).max of the primary tumor area and abnormal activity in the larynx and neck lymph node areas were measured. No particular SUVmax value was used to distinguish between malignant and benign tissue. SUVmax in abnormal uptake areas and on the primary tumor area were measured with an automatic 3D tool or when that was not possible, by drawing a region of interest (ROI), encompassing the whole area with the abnormal uptake. In addition, findings were classified according to the NI-RADS qualitative criteria [Similarly to the original reporting style, imaging studies were re-assessed using a 3-step scale: negative for malignant disease, equivocal, and positive for malignant disease. The interpretation was negative when there was no abnormal uptake or only minimal mucosal linear uptake present. The interpretation was equivocal when focal mild uptake or moderate mucosal linear uptake was observed. The interpretation was positive when there was intense focal uptake, enlarging or a new mass. SUVcriteria . The sofThe patients fasted for six hours and had blood glucose levels < 10 mmols before the imaging. After an intravenous injection of approximately 350–450 MBq (5 MBq/kg) of 18-Fluoride-fluorodeoxyglucose (18 F-FDG) and 60 min of resting, images were acquired on an integrated PET-CT scanner. Body images were obtained separately from head and neck images. CT was imaged from the jugulum to mid-thigh, with the patients’ arms raised above shoulder level. CT parameters were 120–140 kV, 50–60 mAs and a section width was 4 mm. The examination was followed by a PET scan, with an 8 cm bed position and 1.5 min per frame. The head and neck were imaged with the patients’ arms positioned down. CT parameters were 120 KeV, 50 mAs, and a section width of 3 mm. PET had an 8 cm bed position and 2.5 min per frame. CT was performed without contrast, per the institutional protocol. The PET-CT system was Philips Gemini GXL  until 2015, and Siemens Biograph  thereafter. A correction factor was used to transform SUV measures to be comparable between the two systems.max value for optimal sensitivity and specificity was assessed using receiver operating characteristic (ROC) curve analysis. A p-value of ≤ 0.05 was considered statistically significant.The times to recurrence and endpoints were counted from the last date of RT. Follow-up was defined as from the last date of RT to the last clinical visit. We calculated the sensitivity, specificity, PPV, NPV, and accuracy of PET-CT in assessing local residual tumors. Differences in categorical and ordinal variables were compared with a Chi-square test or Fisher’s exact test and in continuous variables with a Mann-Whitney-U nonparametric test. Cox logistic regression and Kaplan-Meier analysis were utilized to calculate association of pre-treatment factors with local residual or recurrent disease within 5 years. The primary tumor area SUVmax value was used in the assessment of malignancy, for statistical purposes, we selected SUVmax>3.0 as an abnormal uptake-variable. The statistical analysis was performed with SPSS .For the statistical analysis, the PET-CT 3-step scale for residual disease was transformed into a two-class variable; we used both sensitive and specific interpretations. In the sensitive interpretation, the equivocal result was interpreted as positive, and in the specific interpretation, the equivocal result was interpreted as negative . AlthougThe patient and tumor data are presented in Table Of 73 patients 10 (14%) had residual disease: 9 in the area of primary tumor, and 1 in regional lymph nodes. The median time from the end of treatment to the diagnosis of primary tumor area residual disease was 4.3 months (range 2.3-6.0). Of 73 patients 12 (16%) had recurrent disease: 11 presented with local, and 1 with regional recurrence. Seven (9.6%) presented with distant metastases, of which 4 were discovered in the post-treatment PET-CT and 3 later during the follow-up. One of these also had local recurrence. Altogether, 18 out of all 20 patients with local residual or recurrent disease (90.0%) underwent salvage surgery; 9 who had a residual tumor, and 9 who had a recurrent tumor.The larynx preservation rate  was 75.3%, and 3-year disease specific survival (DSS) was 83.6%. 3-year DSS in different T-stages was 92.9%, 77.8%, and 75.0%, in T2, T3, and T4a, respectively. DSS between different T-stage categories was not statistically different.In the 5-year univariate Cox logistic regression analysis, pre-treatment vocal cord fixation and primary tumor diameter larger than median 2.4 cm were associated with local residual and recurrent disease. HR for vocal cord fixation was 2.649 , and HR for primary tumor diameter was 2.771 . T-stage 3–4 versus 2 was approaching significance (p = 0.064) for inferior local control. Multivariable analysis  showed no statistically significant prognostic factors.max in negative, equivocal and positive interpretation categories were 3.097, 4.619 and 6.023 (p > 0.001), respectively  developed later recurrence on the primary tumor area. These three recurrences occurred at 7, 9, and 11 months after treatment. Findings in the post-treatment PET-CT could not predict future local recurrence.In 16 (21.9%), the PET-CT interpretation locally was false positive, which included positive or equivocal interpretation, with negative biopsy results. There were no patient, treatment or imaging variables including the time point of post-treatment imaging that were statistically significantly associated with false positive interpretations compared to real residual disease.The sensitivity, specificity, PPV, and NPV of the PET-CT in assessing local residual tumors did not differ significantly between patients scanned over 3 months after treatment compared with those scanned at 2–3 months after treatment.max in primary tumor area residual disease were 6.267 and 6.360 (range 4.060–8.880), respectively, and in non-residuals, 3.508 and 3.115, respectively (range 0.940–8.380) (p < 0.001)  false positive interpretations had SUVmax over 4. Thus, the SUVmax values overlapped. The remaining mass on the primary tumor area existed in 5 out of 9 (56%) of the local residuals, and 15 out of 64 (23%) of the non-residuals (p > 0.05). When SUVmax over 4.0 was combined with residual mass, specificity and NPV rose to 91% and 94%, respectively. None had developed a new or increased mass on the primary tumor area.The mean and median SUVmax of the primary tumor area were 3.7, 4.7, and 4.2, respectively, and all had a persistent mass on the CT. The diagnosis of recurrence may have been delayed 5 months, 3.5 months, and 8 months, respectively, because of the initial false negative biopsy .Vocal cord uptake in non-residuals was found in 32 out of 58 (55.2%). Of these, 7  were on the area of no previous tumor growth, and 25  on the area of previous tumor growth. Most of these were asymmetric (90.6%), and associated with a false positive PET-CT interpretation (p = 0.001). Arytenoid cartilage area uptake was common in both groups , and not associated with residual tumors or false positive interpretations. In patients with no residual disease and no previous tumor growth on the arytenoid uptake area, bilateral uptake was present in 31  and unilateral uptake in 6 . Patients with broad, symmetric, artifact-like uptake were removed from this analysis.max over 4.0 and a persistent mass present at the primary tumor area. Our study revealed better sensitivity and NPV compared to the results of a recent review consisting of 3 studies comprising mostly laryngeal carcinoma patients [max thresholds are used.In our study, consisting of only laryngeal carcinoma patients, the NPV of PET-CT at the primary tumor area was extremely high. Thus, all patients presenting without a persistent mass, and either a minimal uptake or no uptake, had a complete local response. In assessing residual disease, however, equivocal and positive PET-CT findings are less reliable; the positive predictive value was the highest (only 46%) with SUVpatients . Anotherpatients . They remax cut-off points have been used in previous studies for post-treatment HNC, but the consensus is lacking, probably because malignant and benign findings considerably overlap [max measures metabolic rate, and it may be high in various inflammatory conditions, and low in malignant tumors that are small, and have a low metabolic rate, or because of the patient’s hyperglycemia. Murakami et al. showed SUVmax over 3.0 to be a usable threshold in malignant neck nodes over 15 mm in diameter [max over 4.0, but 28% of the non-residuals also surpassed this threshold. In a study by Slevin et al., all local laryngeal residual tumors that had SUVmax reported, showed SUVmax over 4.0 [max cut-off value of 3.35 to achieve a sensitivity estimate of 93.75% and a specificity estimate of 91.67%, but the results are not completely comparable to ours as the primary treatment modalities included surgery [max for individual imaging studies were not specified or it remained unclear whether the tumor was residual or recurrent [Many different SUV overlap , 13, 14.over 4.0 . Oe et a surgery . In anotecurrent , 18. Witmax values overlapped with those with residual tumors, which understandably was associated with false positive interpretations , and SUV3%, and Sons Fig. .max over 4.0 and mass were combined, specificity and NPV were high, so this combination usually indicated the presence of residuals. The absence of mass, however, could not rule out residual disease. Three patients in our cohort exhibited elevated SUVmax, persistent mass, and positive PET-CT interpretation, but biopsies were negative. All had local recurrence soon after. In this kind of situation, negative biopsy results should be regarded with suspicion.Although the mass on CT is used in NI-RADS classification, we could not find studies that examine this imaging marker’s effectiveness in post-treatment laryngeal carcinoma. We hypothesized that it would help in differentiating residuals from post-treatment changes. The mass alone proved to be more common in residuals but also occurred relatively often in non-residuals. When SUVA direct laryngoscopy of a suspected residual tumor requires general anesthesia and does not always diagnose the residual tumor correctly. Biopsies may complicate healing and make the interpretation of subsequent imaging more difficult. Comparatively, in a study with 131 radiotherapy-treated laryngeal carcinomas, the false negative rate regarding biopsies of suspected recurrence was 31% . RepeateTwo studies including various HNCs, laryngeal cancers being the minority, found out that in the repeated PET-CT one month or later after the initial post-treatment equivocal PET-CT, the conversion to a complete response rate was 48%  and 60% MRI that includes diffusion weighted imaging (MR-DWI) has also shown promise in differentiating laryngeal or hypopharyngeal residual tumors, with one series achieving a sensitivity of 94% and a specificity of 100% . We coulIn our univariate analysis of pre-treatment prognostic factors, we found that a larger primary tumor diameter and vocal cord fixation were positively correlated with local residual or recurrent disease. Primary tumor size, measured as volume, was found to have strong prognostic value in a recent systematic review and meta-analysis examining imaging factors associated with laryngeal or hypopharyngeal carcinoma recurrence after CRT . This fiOur study has the advantage of including over twice the number of patients compared with previous studies including only laryngeal carcinoma patients , 18. TheDefining a time threshold for residual disease too close to the post-treatment imaging may lead to a false negative scan to be interpreted as a true negative, if biopsy confirmation of a relapse is done after the residual time threshold has passed. This would potentially increase the sensitivity of the imaging test. In our cohort there was one patient with early recurrence and with negative PET-CT. The routine post-treatment PET-CT was performed 3 months after treatment and the suspicion of the recurrence arose 6 months later, confirmed by focal strong uptake at PET-CT, and positive biopsy. Biopsy was not taken after the initial negative scan so it cannot be known for sure if this was indeed a residual, but the time interval between the scan and the recurrence diagnosis seems sufficient to condemn this event as a recurrence and not a residual. Negative PET-CTs without a clinical suspicion are not confirmed with biopsies, which leads to a phenomenon called partial confirmation bias. This potentially could increase sensitivity and decrease specificity. It is a common problem in diagnostic test study designs.Repeated imaging with PET-CT or MR-DWI instead of laryngoscopy and biopsies in equivocal and positive cases could be a feasible future research topic. Also, the ability of a combined PET-MRI scan to find recurrence, specifically in laryngeal cancer, remains to be explored. Another worthwhile topic is to investigate in a large patient cohort whether delaying the first post-treatment imaging to 4 or 5 months would produce more accurate results, and help to reduce false positive interpretations.max values and common focal uptake in the primary tumor area in non-residuals. All residuals had SUVmax over 4.0. Combining this factor with a presence of mass in the CT increased specificity, but this combination could not be used as the sole deciding factor, as its sensitivity was low.In our study, the negative interpretation was reliable, but equivocal and positive results have low PPV and require further diagnostics. Local residual was as common in the equivocal interpretation category as in the positive interpretation category. Interpretation is complicated by overlapping SUV


COVID-19 is a universal challenge and novel disease is one of the core public health concerns. This study aimed to determine the factors that affected COVID-19 prevention behaviors (CPB) based on constructs of Social Cognition Theory (SCT).t-test, and multiple linear regressions. The significance level of α = 0.05 was considered for all tests.This cross-sectional study was conducted on 426 participants referred to health centers of Arak from October 2021 to February 2022, and they were selected through a multi-stage random sampling method. Data were collected via a self-administered questionnaire which includes socio-demographic data (6 items), COVID-19 prevention behaviors (12 items), and SCT constructs related to COVID-19 (32 items). SPSS Ver-16 statistical software was used to analyze the data with one-way ANOVA, independent samples p < 0.001), self-regulation , and self-efficacy , age, gender, and history of COVID 19infection were associated with CPB and those described 61% of the variance of CPB.The mean age was 37.8 years (SD = 12.2) and ranged from 17 to 81. According to the results, 51.2% had higher education and 52.6% of the participants were female. The mean (SD) of COVID-19 preventive behaviors was 43 (SD = 7.8) out of 60. There was statistical association between CPB and three constructs of SCT. Multiple regression showed that the outcomes expectations (β = 0.11, Rendering to the result of this study constructs of SCT is the key predictor of participants’ CPB. Hence, based on these predictors, effective interventions and healthy messages could be designed based on this predictor—outcomes expectations, self-regulation, social support, and self-efficacy which can be beneficial to improve healthy behavior. A new contagious disease was reported in December 2019 and the World Health Organization (WHO) called it COVID-19 on 30 January 2020 [Toward prevention and surveillance of COVID-19 WHO has delivered numerous guidelines about this situation. It spreads generally from person to person via respiratory droplets, sneezing, cough talk, shouting, or singing . LiteratSociologists and psychologists have recommended the use of theories and models to explain the factors affecting health behaviors. Behavioral theories help to better understand the complexity of the factors affecting health behavior and reinforcing educational interventions , 12. StuWe will not need a theory if we know the rate of COVID-19 or the rate of adherence to COVID-19 prevention behaviors. However, when we want to know the impact of various factors on the adoption of healthy behavior, such as why people do not wear masks or why they do not follow COVID-19 prevention behaviors, we will need to use behavioral theory to better explain and understand it.With considering the difference in numerous reports as well as the less use of social cognitive theory studies and the need to investigate the behavior of the people of a community regarding infectious respiratory diseases in the event of outbreaks similar to the Covid-19 disease in the post-corona era were pointed out as existing information gaps in introduction section. Therefore this study aimed to determine the factors affected by COVID-19 prevention behaviors (CPB) based on constructs of the SCT.This cross-sectional study was directed at 426 participants who were referred to the health centers of Arak, from August 2021 to November 2022. Arak is located in central Iran and the center of Markazi province a population of around 600,000.The sample size was considered 430 based on a 40.9% prevalence of COVID-19 prevention behaviors established in a previous study, with a level of confidence (zα/2 = 1.96), 5% level of precision, and non-response rate (estimated to be 20%). Using the following formula:The multi-stage stratified method was used for sample selection. Two health centers were randomly selected from five regions according to socioeconomic status. Then, at each health center eligible participants were randomly selected finally, 426 eligible participants were selected from 10 health centers from five districts of Arak.The inclusion criteria were willingness to participate in the study and not suffering from special mental and emotional diseases.Data were collected by a self-administered questionnaire which includes socio-demographic data (6 items), COVID-19 prevention behaviors (12 items), and SCT constructs related to COVID-19 (32 items). Data were collected through self-administered and completing the questionnaire took nearly 20 min. The strengthening of the reporting of observational studies in epidemiology (STROBE) statement was applied in this study.Socio-demographic data comprised gender, age, education level , occupation and marital status, and economic status .The COVID-19 prevention behaviors were measured with 12 items. For example, the frequency of mask use was asked and the given score ranged from 1 (never) to 5 .The SCT constructs related to COVID-19 were assessed through 32 items derived from the available literature according to the SCT procedures and guidelines , 19. TheThe content validity of measuring tools was established by the expert panel of ten academicians in this field . The content validity ratio (CVR), content validity index (CVI), and reliability . The purpose of the study was explained to the participants, and written consent was then obtained from participating mothers who volunteered to enter this study. Informed consent from illiterate participants were completed through face-to-face interviews at each health centre.t-test, and multiple linear regressions. The significance level of α was considered 0.05 for all data. Data were analyzed by SPSS 16 using.Data were analyzed through SPSS version 16  statistical software using one-way ANOVA (with Bonferroni post hoc method), independent samples p < 0.05) were statistically associated with CPB. Moreover according to the post hoc Bonferroni test, the average of preventive behaviors of Covid-19 is higher in people with over than 75 years old, people with higher socio-economic statuses and higher education, and people with higher self-regulation (p < 0.001).The mean age was 37.8 years SD = 12.2)and ranged from 17 to 81. Results showed that 52.6% (224/426) of the participants were female and 51.2% (281/426) had higher education. The mean (SD) of COVID-19 preventive behaviors was 43 (SD = 7.8) out of 60. About 241 participants (56.6%) had adequate self-regulation , participants’ adequate self-efficacy was 60.3% (257/426) (95% CI = 39. 9% ‒79.6%), and 394 participants (92.5%) had intermediate outcomes expectations (95% CI = 63. 8% ‒98. 8%). Table .2and ranp < 0.001), self-regulation , and self-efficacy , age, gender, and history of COVID 19infection were associated with CPB and those described 61% of the variance of CPB  Table . MultiplThis study discovered the association between SCT constructs and COVID-19 preventive behaviors (CPB). To our knowledge, few documents have been published about the CPB via STC constructs.In the present study, the self-regulatory (SR) structure influenced the participant’s CPB and the mean score of CPB among participants with adequate SR status was significantly higher than that of poor SR (47.2 vs 30.2). Lin et al.’s study , aimed tIn a study Lauren et al.  about baAnother result of this study was the positive effect of outcome expectations on adopting CPB. It can be said individuals who had an intermediate level of outcome expectations had a higher score CPB compared to people who had limited outcome expectations (43.7 vs. 34.3), which confirms the previous studies by Cavicchiolo et al.  and SiniIn a study by Coroiu et al.  about baA study by sinicrope et al. showed that the perceived outcomes expectations toward using a mask are effective on the behavior of using a mask . Also, tIn a study about COVID-19 vaccine acceptance and hesitancy in low- and middle-income countries indicated that messages highlighting vaccine efficacy and safety, delivered by healthcare workers, could be effective for addressing any remaining hesitancy in community . Hence, R = 0.55, P < 0.001). This result is reliable with previous studies that found a significant association between self-efficacy and COVID-19 prevention behaviors [This study displays that COVID-19 prevention behaviors were significantly and positively associated with participants’ self-efficacy towards COVID-19 prevention behaviors  on participants’ COVID-19 preventive behaviors. These results are consistent with some studies. In the study by Raude et al.  which waIn this study the almost participant use outdoor face mask, maintaining physical distance and avoid of crowded areas and social events. Also, this finding is similar to other studies , 10, 29.There were some limitations in this study. First, the questionnaire was self-reported which could be subject to recall and response bias. Second, this study, might not lead to the casual interpretations of constructs of SCT and CPB. Despite limitations, the seemly, valid, and cost-effective method of data collection is self-assessment on adolescents and adults. This study also can create some other scientific evidence for future studies about health promotion communities.Rendering to the result of this study constructs of SCT is the key predictor of participants’ CPB. Hence to design useful and effective behavioral change interventions based on evidence in the field of COVID-19 prevention. Finally based on these predictors, effective interventions and healthy messages could be designed based on this predictor outcomes expectations, self-regulation, social support, and self-efficacy which can be beneficial to improve healthy behavior.


The risk of cardiovascular disease (CVD) depended on the magnitude and exposure duration of insulin resistance (IR). This study aimed to investigate the associations of cumulative metabolic score for IR (cumMETS-IR) with incident CVD, and to further explore the modulated effects of time course of METS-IR accumulation.We enrolled 47,270 participants without CVD and underwent three examinations during 2006–2010 from the Kailuan study. CumMETS-IR from 2006 to 2010 were calculated as the mean values of METS-IR between consecutive examinations multiplying by time intervals between visits. Time course of METS-IR accumulation was calculated as the slope of METS-IR versus time. Hazard ratios (HRs) and 95% confidence intervals (CIs) for CVD risk were calculated with multivariable-adjusted Cox regressions.During a median follow-up of 10.99 years, we identified 3184 cases of incident CVD. The risk of incident CVD increased with increasing cumMETS-IR , exposure duration , and cumulative burden . A positive slope was associated with 14% higher risk of CVD . When combining cumMETS-IR and slope, those with cumMETS-IR ≥ median (142.78) and slope ≥ 0 had the highest risk of CVD .The risk of CVD increased with elevated cumMETS-IR and an increasing trend over time, emphasizing the importance of maintaining optimal METS-IR levels across life span.The online version contains supplementary material available at 10.1186/s12933-023-02073-2. Insulin resistance (IR), which refers to the diminished or impaired insulin sensitivity of target organs or tissues shown as impairments in absorbing and oxidizing the glucose , 2, has Considering the practicality of its measurements and the pathophysiological correlations with components of metabolic syndrome and IR, a predictive role of METS-IR has also been highlighted in endothelial dysfunction and inflammatory , 12. MorTherefore, based on a large cohort study, we aimed to quantify the association of (1) cumulative METS-IR (cumMETS-IR); (2) exposure duration of high METS-IR; (3) cumulative burden of METS-IR with the risk of CVD, and further to assess whether the associations were modulated by time course of METS-IR accumulation.The participants were recruited from the Kailuan study, which was an ongoing prospective cohort study conducted in Tangshan, China. Details of the study design and procedure have been described previously –21. From2). Fasting blood samples were collected in the morning after an 8- to 12 h overnight fast. All the.Information on demographic characteristics, lifestyle, and medical history was collected though face-to-face interview via a standard questionnaire. Height, weight, and blood pressure were measured by professionally trained doctors. Body mass index (BMI) was calculated as weight divided by height squared  at the central laboratory of Kailuan Hospital, including fasting blood glucose (FBG), lipid profiles , serum creatinine, and high sensitivity C reactive protein (hs-CRP). Estimated glomerular filtration rate (eGFR) was calculated using Chronic Kidney Disease Epidemiology Collaboration creatinine Eq. 22The equation for METS-IR calculation was as follows :\documenCumMETS-IR was defined as the summed average METS-IR for each pair of consecutive examinations multiplied by the time between these two consecutive visits in years:2006, METS-IR 2008, METS-IR 2010 indicated METS-IR at baseline, the second examinations (2008), and the third examination (2010), time2006-2008 and time2008-2010 indicated the participant-specific time interval between consecutive examinations in years /2–cutoff] × time2006-2008 + [(METS-IR 2008 + METS-IR 2010)/2-cutoff] × time2008-2010. If the values of cumulative burden were less than 0, this value would be considered as 0.Where METS-IR Time course of cumMETS-IR accumulation was calculated as a slope of METS-IR over time from 2006 to 2010 using the linear regression and the least-squares principle, where METS-IR was taken as the dependent variable, and time from 2006 to 2010 as the independent variable, with a positive or negative slope indicating an increase or decrease in METS-IR over time . The primary outcome in the study was incident CVD, including incident stroke and myocardial infarction (MI). We used ICD-10th revision codes to identify CVD cases . All participants were linked to the Municipal Social Insurance Institution and the Hospital Discharge Register for incidence of CVD, which cover all of the Kailuan study participants and updated annually during the follow-up period. To further identify potential CVD events, we reviewed the discharge lists from the 11 hospitals during 2006–2021 and asked for a history of CVD via a questionnaire during the biennial interview. For all suspected CVD events, 3 experienced physician adjudicators who were blinded to the study design reviewed the medical records. The diagnosis of incident stroke was confirmed by medical review, using the World Health Organization criteria . MI was Participants were classified according to quartiles of cumMETS-IR, METS-IR slope (positive or negative), or the combination of median cumMETS-IR with slope, respectively. The optimal cutoff point for METS-IR associated with incident CVD was determined using an outcome-oriented method to maximized log-rank statistics .Baseline characteristics were compared using student t test, analysis of variance, Wilcoxon, or the Kruskal–Wallis test according to distribution, and categorical variables were compared with chi-square test. Kaplan–Meier curves were used to estimate the cumulative incidence of CVD and the differences in curves were compared with the log-rank test.Multivariable-adjusted Cox proportional hazard regressions were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for the risk of incident CVD. Three models were constricted progressively. Model 1 was adjusted for age and sex; model 2 was further adjusted for education, income, smoking status, drinking status, and physical activity; and model 3 was further adjusted for history of hypertension, diabetes, dyslipidemia, total cholesterol, eGFR, and hs-CRP. The proportional hazards assumption was satisfied by checking the Schoenfeld residual plots. Restricted cubic splines adjusted for variables in model 3 were performed to capture the dose–response relationships of cumMETS-IR and METS-IR slope with the risk of CVD, with 4 knots at the 5th, 35th, 65th, and 95th percentiles of the distribution according to Bayesian information criterion and Akaike information criterion.2), FBG (≥ 126 mg/dL), TG (≥ 150 mg/dL), and HDL-C (< 38.66 mg/dL). Additionally, subgroup analyses stratified by age (< 60 years vs ≥ 60 years), sex (women vs men), BMI (< 24 kg/m2 vs ≥ 24 kg/m2), FBG (< 126 mg/dL vs ≥ 126 mg/dL), TG (< 150 mg/dL vs ≥ 150 mg/dL), and HDL-C (< 38.66 mg/dL vs ≥ 38.66 mg/dL) were performed, interaction between subgroups were tested using likelihood ratio tests, in which models with and without multiplicative interaction terms were compared.Several sensitivity analyses were performed to validate the robustness of the results. First, the competing risk model was performed by considering non-CVD death as a competing risk. Second, to minimize the potential impact of reverse causality, we repeated the primary analysis using a 1 year lagged period by excluding participants who developed CVD cases within the first 1 years of follow-up. Third, restricted analysis was performed by excluding participants with abnormal BMI . All the statistical tests were 2-sided, and A comparison in baseline characteristics between excluded and included participants is presented in Additional file P < 0.0001). This trend remained significant even after adjustment for potential variables, the HR for the risk of incident CVD was 1.38 (95% CI 1.23–1.54), 1.44 (95% CI 1.29–1.61), and 1.77 (95% CI 1.58–1.98) for the Q2, Q3, and Q4 versus the Q1 group of cumMETS-IR , a total of 3,184 cases (6.74%) of incident CVD occurred, including 2,614 cases (5.53%) of stroke and 626 cases (1.32%) of MI. The incidence rate of CVD increased substantially with increasing cumMETS-IR, ranging from 3.91 (95% CI 3.58–4.27) per 1000 person-years in the Q1 group to 9.30 (95% CI 8.77–9.87) per 1000 person-years in the Q4 group, which was also illustrated in Additional file Using an outcome-oriented method to maximize log-rank statistics, the optimal cutoff point of mean METS-IR associated with CVD was ≥ 33.61 Fig. . With thAssociations of the time course of METS-IR accumulation with the risk of incident CVD were presented in Table P > 0.05 for interaction; Additional file Sensitivity analysis with competing risk, excluding incident CVD within 1 year, and restricting participants with normal BMI, FBG, TG, and HDL-C yielded similar results  of METS-IR over time conferred a higher risk of CVD. Individuals with increasing trend, even the levels of METS-IR decreased afterwards, still had a higher risk of CVD. Additionally, incorporation of both cumMETS-IR and the time course, the results showed that same cumMETS-IR exposure accumulated with an increasing trend compared with a decreasing trend contributed more to the subsequent risk of CVD. The results indicated that an increasing in METS-IR over time, even from a relatively lower level to result in the same accumulation, did not fully decrease the risk acquired progressively. Possible reasons may be that atherosclerosis caused by increasing METS-IR, is a chronic progressive disease that begins early in life and develops over the course of decades before becoming clinical manifestation .Although the precise mechanisms linking cumMETS-IR and CVD risk remain incompletely understood, several potential interpretations have been proposed. First, due to the involvement of BMI, METS-IR might be a better indicator of IR in adipose tissue, muscle and liver . TherefoThere are some interesting implications of this study, especially when viewed in the context of other studies. First of all, the assessment of CVD risk is informed by considering not just the total amount of METS-IR, but also the time course of the accumulation. In current practice, the METS-IR at the time is used without trying to incorporate the modulation of that risk by the time course of the individual’s METS-IR levels. We developed a risk model that took into account both of these descriptors of longitudinal METS-IR exposure. The results demonstrated here both emphasized the dependence of risk of CVD, not just on the present METS-IR levels, but also the time course of accumulation, and offer a model to quantify the modulation of risk by the time course. the clinical application of METS-IR. These data suggested that prolonged exposure to lower METS-IR, beginning early, is contributed more to the risk reduction of CVD.The strengths of our study included the large sample size with a long follow-up, and the components of METS-IR were measured repeatedly. Additionally, we used cumulative value of METS-IR to capture the longitudinal exposure of METS-IR, and incorporated both cumulative exposure and time course of METS-IR accumulation into one risk parameter to predict future CVD, which conferred additional information beyond a single measurement of METS-IR. However, several limitations should also be noted. First, insulin concentrations were not collected in our study due to the large population with high cost, we could not compare the predict value of cumMETS-IR with the cumulation of the gold-standard for the risk of CVD. Second, owing to the observational nature of the study, we could not establish a causal association of cumMETS-IR with the risk of CVD. Third, residual confounding cannot be completely ruled out due to the limitation of observational study design, despite comprehensive adjustment for the potential confounders. Finally, the sex distribution of the sample was unbalanced. However, the associations were statistically robust, given that a significant interaction was not identified when data were stratified by sex.Incident CVD risk was associated with both long-term exposure to METS-IR and the time course of METS-IR accumulation. Importantly, the same cumMETS-IR with an increasing trend resulted in a greater risk increase, emphasizing the importance of control an optimal METS-IR across the lifespan.Additional file 1: Table S1. Baseline characteristics of excluded and included participants. Table S2. Association of cumulative exposure to METS-IR with the risk of stroke. Table S3. Association of cumulative exposure to METS-IR with the risk of myocardial infarction. Table S4. Association of time course of cumulative METS-IR with the risk of stroke. Table S5. Association of time course of cumulative METS-IR with the risk of myocardial infarction. Table S6. Sensitivity analyses for the association of cumulative METS-IR with the risk of cardiovascular disease. Table S7. Sensitivity analyses for the association of time course of cumulative METS-IR with the risk of cardiovascular disease. Table S8. Subgroup analyses for the association of cumulative METS-IR with the risk of cardiovascular disease. Table S9. Subgroup analyses for the association of time course of cumulative METS-IR with the risk of cardiovascular disease. Figure S1. Kaplan-Meier curves of cardiovascular disease and its subtypes incidence rate by quartiles of cumulative exposure to metabolic score of insulin resistance. Figure S2. Hazard ratios and 95% CIs for the association of cumMETS-IR and METS-IR slope with the risk of stroke by using restricted cubic spline regression with 4 knots with placed at the 5th, 35th, 65th, and 95th percentiles. Figure S3. Hazard ratios and 95% CIs for the association of cumMETS-IR and METS-IR slope with the risk of myocardial infarction by using restricted cubic spline regression with 4 knots with placed at the 5th, 35th, 65th, and 95th percentiles.


The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease “virtually” incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution  of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte  and macrophage -associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan–Meier estimates were linked with shorter EFS , with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts. Follicular Lymphoma (FL) represents the most common type of indolent non-Hodgkin lymphoma, accounting for about 7–15% of all lymphomas worldwide . The twoEZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP and CARD11. Despite its effectiveness, this specific type of individual genetic evaluation is not practical in the clinicopathological setting [The FLIPI  clinical score and its evolution to FLIPI2, also applicable to rituximab-treated cases, are widely used in clinical practice to categorize patients in risk groups  . These s setting . The mos setting ,12. Tumo setting ,14, CD4+ setting  are the  setting . Dave etA total of 49 FL patients (26 men and 23 women) with sufficient formalin-fixed paraffin-embedded (FFPE) material were included in the study. Overall, the clinical and pathological characteristics of the patients were well-balanced . The hisp-value 0.94; p-value 0.96; p-value 0.02; p-value 0.04; p-value 0.003; EF: p-value 0.002; p-value 0.48; EF: p-value 0.93, p-value 0.01; A pathologist-guided analysis of CD68 (PGM1 clone) marker expression was conducted on FL lymph node biopsies collected at the time of diagnosis. To this aim, standard IHC followed by whole-slide-image (WSI) acquisition was performed . CD68 pop-value 0.03; p-value < 0.001; Given the macrophage plasticity across cancers, we decided to further characterize the functional polarization of FL macrophages utilizing CD163 as a broadly applied marker of tumor-associated myeloid cells. Sequential IHC , Panel ABCL2 positivity . Analogous results were recorded for CD56/MS4A4A EF ratio, where values lower than 18/HPF were associated with better EFS . With regard to CD68 abundances, CD68 IF lower values were associated with worse EFS , while no effect was detected for CD68 EF . No baseline clinical variables showed evidence of association with EFS. Multivariate analysis based on markers that were significantly correlated with EFS did not reveal any independent effect  . Italy aFurthermore, POD24, even if an association with an inferior OS has been validated, requires two years’ evaluation and the administration of a first unsuccessful line of therapy ,27. InflIn our study, the prognostic effect of CD68+CD163- cells restricted to the CD21+ FDC distinctive meshwork staining pattern was only emphasized by the compartmentalized analysis of IF vs. EF areas. Our evaluation was carried out on the samples collected at the diagnosis and, even if most of the patients in our cohort (43/49) received rituximab as a first-line treatment, it emerged that the macrophage component is the one that has an impact on the patient’s prognosis. It was beyond the scope of this study to define whether the presence of rituximab in the therapeutic regimen counteracts a possible negative effect of the CD68+ subset within the germinal center or whether it performs a synergistic action with it in the elimination of the pathological elements. Nevertheless, taken together, our data underscore the relationship between IF CD68+ cells in the FL microenvironment and clinical outcome as opposed to those localized in EF areas, which did not appear to influence the disease course.p-value (0.06) is close to statistical significance, we assume that this is the case. Moreover, IHC examination revealed that CD163 immunoreactivity was mainly absent within the follicles, although MS4A4A+ cells were present, demonstrating the capacity of MS4A4A+CD68+ macrophages to infiltrate the follicles and influence the course of the disease.Further characterizing the myeloid component, we found that EF areas were enriched with FL-associated macrophages expressing CD163 and/or MS4A4A markers. Moreover, in line with previous reports , these mIt has to be acknowledged that the genome-wide approach gave us many insights into FL microenvironment biology ,12. Davep = 0.0469)  . To iden 0.0469) . HoweverIn summary, our analyses showed that FL immune subsets inside and outside the follicles have a diverse impact on EFS. This emphasizes how crucial it is to separately analyze IF and EF areas. Certainly, digital spatial profiling techniques, in use at the research level, might fill this gap, but are not suitable in terms of time and cost to be incorporated into the diagnostic laboratories and for the frontline management of patients. We believe that our proposed IHC subset analysis, including CD163 MS4A4A and CD68 for macrophage and measuring their level with respect to specific lymphoid markers, can aid in the prognostic stratification. As previously mentioned, all patients in our cohort except for six received rituximab-based systemic therapy as a first-line treatment. Validation of our results was also separately conducted performing a sensitivity analysis to assess the potential impact of surgery-treated patients on marker’s effect. The observed differences on EFS according to CD68 IF, CD163/CD8 EF, CD56/MS4A4A EF and PD1 IF values remained robust to the exclusion of the only surgery-treated/wait-and-see patients . This woThe study was approved by the Local Ethical Committees of Area Vasta Romagna (CE 194/2021/Oss/AOUBo) and “L. e A. Seràgnoli” (Bologna) and informed consent was obtained from each patient for their biological material to be used for research purposes. FL classification, including histopathology and immunophenotyping, was performed at the Institute of Hematology “L. e A. Seràgnoli” according to the World Health Organization classification [Tissue samples obtained through surgery were fixed in neutral buffered formalin and embedded in paraffin. Four-micron sections were mounted on positive-charged slides . Immunostaining for biomarkers was performed at the Biosciences Laboratory of IRST IRCCS  using the Ventana Benchmark ULTRA staining system  with the Optiview DAB Detection kit . All used antibodies are listed in For sequential IHC, a non-biotin poly HRP conjugate system followed by aminoethyl carbazole (AEC) substrate reaction was used instead of 3,3-diaminobenzidine (DAB). All the AEC conditions were validated on tissue microarrays (TMAs) containing different positive control tissues or on human tonsils . Each map-value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism  and R version 4.2.0.For continuous variables, the data were summarized by median and range and for categorical variables by frequency and percentage. Statistical analysis was performed using GraphPad Prism . POD24, BCL2, FLIPI and grade were the clinical and biological parameters utilized to compare differences in the number of positive cells for a given marker or ratio using the Mann–Whitney U. Regarding Stage, Kruskall-Wallis test was used to evaluate the association with each given marker or ratio and Dunn’s multiple comparison test was used for the post hoc test. The Kaplan–Meier method was used to estimate EFS defined as the time from diagnosis to first documented progression or relapse of the disease. A log-rank test was used for curve comparisons. Cut points were determined using a recursive partitioning algorithm to stratify each marker or ratio in relation to EFS. The Cox proportional hazard regression model was used to quantify the association between variables and EFS. Multivariate analysis was performed with purely explorative intent to assess the independent effect of each of the variables associated with EFS in univariate analysis. The results are reported as HR and 95% CI. Overall, a two-sided


Cytotoxicity was determined by the MTT-assay, genotoxicity by using the p53-CALUX and Umu-assay, and estrogenic effects by the ERα-CALUX. Genotoxic as well as estrogenic effects were detected in different samples depending on the material and the irradiation type. In four leachates of 12 plastic species estrogenic effects were detected above the recommended safety level of 0.4 ng 17β-estradiol equivalents/L for surface water samples. In the p53-CALUX and in the Umu-assay leachates from three and two, respectively, of 12 plastic species were found to be genotoxic. The results of the chemical analysis show that plastic material releases a variety of known and unknown substances especially under UV radiation, leading to a complex mixture with potentially harmful effects. In order to investigate these aspects further and to be able to give recommendations for the use of additives in plastics, further effect-related investigations are advisable.The broad use of plastics and the persistence of the material results in plastic residues being found practically everywhere in the environment. If plastics remain in the (aquatic) environment, natural weathering leads to degradation processes and compounds may leach from plastic into the environment. To investigate the impact of degradation process on toxicity of leachates, different types of UV irradiation  were used to simulate weathering processes of different plastic material containing virgin as well as recyclate material and biodegradable polymers. The leached substances were investigated toxicologically using  There are only rough estimates for the annual global input of plastic into the environment. Scientists estimated that in 2010, 4.8–12.7 million tons of plastic entered the world’s oceans. They predicted an increase by an order of magnitude until 2025 . Howeverhttp://www.plastrat.de) funded by the German Federal Ministry of Education and Research (BMBF) focused, among other topics, on possible toxicological effects of plastic associated substances. Emphasis was put on the leaching of plastic associated substances after artificial weathering. The effects of leachates from different plastic species (and different weathering scenarios) as well as additives and identified substances from leachates have been investigated and were partly already published by In order to analyze this risk, the “PLASTRAT”-project . The study focused on the impact of UV-radiation on degradation processes, as this weathering scenario was investigated to a lesser extent up to now, especially using in-vitro bioassays for toxicity assessment of leachates. The investigated leachates in the study of via drinking water produced from bank filtration, groundwater and seawater desalination or via sea food and agricultural products. But also food contact materials , clothes  and furniture  are of relevance. With regard to food contact materials, however, there is a EU Regulation (Commission Regulation (EU) 2022/1616) that does not allow to use recycled plastic if not demonstrated to be properly decontaminated. To address all the mentioned points and as additional part of the PLASTRAT-Project, the present study focused on estrogenic and genotoxic effects of plastic-associated substances resulting from different artificial weathering scenarios and thereby complements the study of in-vitro bioassays to assess cytotoxicity (MTT assay) as a pre-test for CALUX assays. Estrogenic as well as genotoxic effects  were investigated. Moreover, the released plastic-associated substances were analyzed in the leachates by high performance liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF-MS). Here, signal intensities and number of components were detected and used to compare the different weathering scenarios with respect to the release of plastic-associated substances. Additionally, substances present in the leachates were chemically analyzed using a non-target approach. To the best of our knowledge, this is the first study investigating cyto-/genotoxic and estrogenic effects of substances released from plastic material under weathering conditions.Thus, we investigated seven petroleum-based plastic species, three recyclates as well as two biodegradable and fully or partly bio-based materials. For each plastic in total four leachates were produced, which underwent artificial weathering, including ‘atmospheric’ and ‘aquatic’ scenarios. Leached chemicals were enriched using solid-phase-extraction and investigated using Selected plastic polymers , artificial weathering and solid phase extraction (SPE) were identical as published by LDPE-R is a post-industrial recyclate from the packaging industry. It is mechanically recycled plastics and contains additives in contrast to the additive-free LDPE.2/L−1) of the decomposed plastic material are given in aq.) were analyzed and are presented in Artificial weathering and SPE were performed in duplicates by project partners from Goethe University Frankfurt (Germany). The starch blend (SB) was processed later at IWW in the same way . Pellet In short and relevant for the present study: for artificial weathering 100 g of each plastic polymer was treated under four different conditions. The simulated scenarios included ‘atmospheric’ as well as ‘aquatic’ weathering. For ‘atmospheric’ weathering conditions, the test materials were irradiated under laboratory conditions for 24 h by either UV-C at 250 nm wavelength  or UV-A/B at 280–400 nm wavelength . To represent an ‘aquatic’ exposure scenario and to cover volatile compounds eventually lost under T2 and T3, test materials were leached for 24 h in 1.1 L of ultrapure water during UV-A/B irradiation at 280–400 nm (T4). A corresponding dark control (T1) was treated as T4 without irradiation. Further weathering conditions e.g., temperature, UV-intensities, lamp characterization and calculation of the simulated irradiance time during weathering are described in SPE was performed with 1 L leachates using Telos C18/ENV cartridges  to retain hydrophobic compounds . Loaded α-transfected human osteosarcoma cell line U2-OS  were cultured in Dulbecco’s modified eagle medium with nutrient mixture F-12  and phenol red . The medium was supplemented with 8.0% fetal calf serum (FCS), 0.6% Gentamicin and 1.0% non-essential amino acids . Both cell types were cultured at conditions of 37 °C, 97% humidity and 5% CO2 atmosphere .p53- and ER4 cells in 100 µL medium per well of 96-well-plates. ERα-cells were incubated under the above conditions for 24 h and p53-cells for 48 h to allow adherence and confluent growth before exposure to the respective sample.For CALUX-assays, cells were seeded in DMEM low glucose  medium (free of phenol red supplemented with 4.8% stripped FCS (BDS), 1.6% gentamicin (c.c.pro), and 1.0% MEM-NEAA (c.c.pro), at a density of 1 × 10The MTT assay is used to determine the metabolic activity of eukaryotic cells and is based on the reduction of the soluble, yellow tetrazolium salt 3--2,5-diphenyltetrazolium bromide (MTT) to a blue-purple, insoluble formazan by dehydrogenases localized in the mitochondria of the cells. After cell lysis, formazan can be quantified photometrically. The amount of formazan is proportional to the number of metabolically active eukaryotic cells.In detail, cells were exposed to different concentrations of the samples for 24 h. 10% DMSO served as a positive control. Subsequently, cells were washed and stained with MTT-solution . After 2 h of incubation, cell lysis  was performed and absorbance at 595 nm was measured using the Spark 10M plate reader .α) and genotoxic (p53) activity  was appactivity .α-CALUX-assay, used for the detection of estrogenic effects was performed according to α). In case an estrogen-active substance binds to the ERα receptor in the cell, the corresponding gene is read and consequently also the reporter gene. Thereupon luciferase is produced, which reduces the added luciferin to oxyluciferin under the emission of light. The luminescence intensity correlates directly with the amount of substance bound to the receptor. To determine estrogenicity equivalent concentrations, a 17β-estradiol (E2) standard series  is included in each assay, which allows the expression of results as ng EEQ/L  and subsequent luciferin  addition. Immediately afterwards, luminescence was measured using a Spark 10M plate reader.post-hoc test  was performed in order to proof if estrogenic effects measured in the plastic leachates were statistically different from the process control.A statistical analysis using One-way ANOVA und Tukey’s α-CALUX-assay. Here, the U2-OS cell line has been genetically modified in such a way that the reporter gene luciferase has been coupled to the tumour suppressor gene p53. The latter is transcripted into proteins after DNA damage and thereby responsible for cell cycle control by regulating other genes involved in apoptosis and DNA repair. If the DNA in a cell is damaged, the translation of p53 starts followed by luciferase production. The luminescence intensity resulting from the reduction of added luciferin is used to indirectly determine the relative induction factor of p53.The p53-CALUX-assay was performed to determine the genotoxic potential in eukaryotic cells and follows the same principle as explained for the ERα-CALUX-assay.Forty-eight hours after seeding in assays medium, adherend and confluent cells were exposed with and without the rat liver extract S9  to account for possible metabolization of the sample ingredients. In the approach without liver extract, cells were exposed for 24 h. For the genotoxicity detection after metabolic activation 20 µL of the S9 mix were added. After 3 h of incubation exposure media containing S9 mix and sample was replaced by assay medium and cells were incubated for another 21 h. Cyclophosphamide (CPA) served as positive control for the approach with S9; actinomycin D (ACTD) was used in the approach without S9 (both Merck KGaA). After an incubation time of 24 h cell lysis, luciferin addition and luminescence measurement were performed analogue to the ERThe fold-induction per well was calculated by dividing the average Relative Light Units (RLU) level of the tested compound by the average RLU of the solvent control DMSO. Actinomycin-D was used as a positive control and tested compounds were considered “positive” when the response of at least one concentration was above the determined 1.5-fold induction threshold and SD <20% .Finally, the IF (induction factor) relative to the negative control was calculated. Plastic leachates were judged to be genotoxic if the IF was greater than or equal to 1.5 in at least two of three test replicates and on average.Salmonella typhimurium TA1535/pSK1002  following DIN standard  to the yellow o-nitrophenol which allows the genotoxic potential of a sample to be determined indirectly by a color reaction at 420 nm. The result is expressed as the induction rate (IR) of the enzyme, which corresponds to the increase in absorbance at 420 nm relative to the negative control. The IR is dependent on the growth rate previously determined at 600 nm. If the growth rate of a sample is less than 0.5 compared to the negative control, the results are disregarded due to cytotoxicity.The Umu-assay was performed with the genetically modified bacterium standard . Genotoxβ-Mercaptoethanol; Sigma, Munich, Germany) and 30 µL ONPG  solution (22.5 mg ONPG in 5 mL Phosphate buffer) for 30 min at 28∘C. Than 120 µL stop reaction solution, containing 53 g disodium hydrogen carbonate per 500 mL distilled water  was added and the extinction was determined photometrical at 420 nm (Spark 10M plate reader). Finally, the induction ratio (IR) relative to the negative control was calculated. Depended on the sample volume in total only two replicates were performed. Calculations of the IR were carried out according to the International Standard ISO 13829 . Bacteria were exposed to the samples for 2 h. Final concentrations of the different extracts tested were 0.67, 1.34, 2.67 and 5.34 g P-EQ /L (SB 0.37 to 2.94 g/L P-EQ). P-EQs are plastic-equivalents based on the amount of plastics used during the leaching experiment in a defined water volume. Exposure of bacteria was performed both with and without the rat liver extract S9 (liver extract Arochlor in KCL) to account for genotoxic effects following metabolization of sample ingredients. As a positive control, 4-nitroquinoline N-oxide (4-NQO) was used in the test batch without S9 and 2-aminoanthracene (2-AA) in the batch with S9 . To determine the growth rate during exposure, 1:10 dilution was used and optical density was measured immediately after 2 h. For detection of genotoxic effects, 30 µL of the treated bacterial solution were suspended in pre-warmed 120 µL B-buffer  and ESI(-) ionization mode (scan mode 100–1200 Da). Samples were diluted 1:1 with ultrapure water and injection volume was 50 µL (The eluates of Solid Phase Extraxtion (SPE) were measured as 50 µL . The datas 50 µL .α- and p53-cells. Test concentrations in the CALUX-assays were chosen based on the results of the MTT-assay.In order to determine cytotoxic effects of the different plastic leachates on eukaryotic cells and to determine the non-cytotoxic concentrations to perform the CALUX assays on estrogenicity and genotoxicity, the MTT assay was performed using ERα-cells: 178 and 445 g/L P-EQ (starch blend pellets (SB) 98 and 196 g/L P-EQ). At the highest concentration tested, cytotoxic effects (<70% cell viability) were measurable in some plastic leachates (see α-CALUX was performed with leachate concentrations of 178 g/L P-EQ (SB: 196 g/L P-EQ), in order to be able to compare the results of the different plastic polymers as well as the different treatments.Two different leachate concentrations were tested in the MTT assay with ERates see . At a coIn the MTT-assay with p53-cells, three different concentrations of the leachates were tested: 445, 890 and 1780 g/L P-EQ . The highest two concentrations showed cytotoxic effects in most of the leachates see . At the α-CALUX was performed with exposure concentrations of the plastic leachates of 178 g/L P-EQ (196 g/L P-EQ for SB). As the procedural blanks (pb) of the SPE using Telos C18/ENV cartridges (Kinesis GmbH) showed slight estrogenic effects (0.29 ng EEQ/L), this background signal was subtracted from results of the leachates, except for SB .The ERThe results of the different treatments are shown in The p53-CALUX was performed with exposure concentrations of the plastic leachates of 445 g/L P-EQ (1958 g/L P-EQ for SB). Plastic leachates were judged to be genotoxic if the induction factor (IF) was greater than or equal than 1.5 in at least two of three test replicates and on average . A genotAs second genotoxicity assay, the Umu-test was applied. Here, plastic leachates were tested in 4 concentrations ranging from 0.67 to 5.34 g P-EQ/mL. (SB 0.37 to 2.94 g/mL P-EQ). Samples were judged to be clearly genotoxic if the induction rate was greater than 1.5 for both test replicates. The results, indicating the lowest genotoxic concentration with or without S9, are presented in The LDPE-R leachates were genotoxic after every type of weathering scenario and after “aquatic” weathering with UV-A/B (T4) already at the lowest tested concentration of 0.67 g/mL P-EQ. The leachates of the weathered SB also exhibited genotoxic potential after T1, T2, and T4. A genotoxic effect after metabolization of substances present in the leachates occurred only for LDPE-R in the dark control (T2).For comparison of the biological activity of the different investigated plastic leachates, a heat map was created based on the bioassay results of this study . The heaAs already discussed in LDPE-R, both biodegradable plastics and both PVC samples released the highest number of compounds  and 4873 , see Tab. S3). PP-H released the fewest number of compounds (between 37 (T2) and 78 (T4); Tab. S3). A clear increase of counts caused by UV irradiation could be observed for LDPE-R and PVC-A. For Bio-PBS and SB only a small effect and for the other plastics no effect could be observed.This study aimed to investigate if chemicals present in plastics are increasingly released after UV-weathering and if the released chemicals can be interlinked with the observed toxicological effects. Therefore, 12 plastic species of PP, PET, PS, LDPE, PVC, Bio-PBS and SB materials were artificially weathered by UV-C, UV-A/B, UV-A/B irradiation in water and a dark control. Our results demonstrate that leachates of plastic materials can exhibit detectable cytotoxic and genotoxic effects as well as endocrine activity.The number of component counts determined under the simulated weathering conditions increased for most of the samples. Only the SB and LDPE-R samples leached fewer chemicals after each UV treatment or solely after ‘atmospheric’ weathering, respectively. As already pointed out by Light stabilizers and other additives were not separately measured. Also, it should be considered when analysing the study results that plastic materials can be produced following different manufacturing processes, using different conditions and formulations. This may result in differences in the leachate profiles and in its toxicity following UV irradiation or environmental degradation.It was beyond the scope of our study to identify the main drivers for the observed toxicities because this would require an effect-directed analysis. We have not primarily focused on identifying chemicals, but future studies should consider this to identify unknown substances that are responsible for adverse effects. A specific identification of the plastic-associated chemicals also went beyond the scope of our study. However, we were able to identify several components see , includihttp://www.plastrat.de) our study complements the investigations of α-CALUX, the p53-CALUX and the Umu-assay.As part of the BMBF-funded PLASTRAT project  recommends a biotest battery when evaluating genotoxic substances in drinking water . Followiow e.g., .One interesting observation in our study was, that all investigated leachates did not show genotoxic effects in p53-CALUX when S9-mix was added. Without S9-mix leachates of PS-HI, LDPE-R and SB showed positive results. A possible explanation could be a metabolic transformation and detoxification of the substances in the leachates by the liver extract (S9-mix). The metabolic transformation of substances by liver enzymes is described in α-CALUX assay performed in this study is based on the binding of substances to an estrogen receptor. In addition to receptor-dependent processes, there are also receptor-independent processes that can affect endocrine functions. These include, for example, the inhibition of enzymes that are responsible for the production, conversion or elimination of steroid hormones. As a receptor-independent test, the steroidgenesis assay, which determines the effect on the production of 17β-estradiol and testosterone, can be performed additionally  was used in this study for treatment of the water samples because it was considered as a suitable method to extract estrogen-like compounds from the water phase. However, the use of this type of extraction in relation to water in contact with plastics has been the subject of controversial discussion in recent years. According to Altogether, our results show that plastic material releases a variety of known and unknown substances, leading to a complex mixture of chemicals with potentially harmful effects. UV radiation can degrade plastic material and can increase release of substances from this material. Environmental impacts and possible human health concerns need further investigations. Not just new but also aged materials should be tested under different weathering conditions. It seems that metabolization of released substances play a role in their toxicological potential. Therefore, metabolization should be considered for risk assessment analyses.10.7717/peerj.15192/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15192/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15192/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15192/supp-4Supplemental Information 4Click here for additional data file.